The study of the response by cytotoxic T lymphocytes (CTL) to minor alloantigens is of interest for a number of reasons. First, it serves as a model for the recognition of tumor and viral antigens by CTL. Second, it is of clinical relevance since minor alloantigens probably serve as the target antigens of graft-versus-host (GVHD) following bone marrow transplantation (BMT) between HLA-identical sibs. Progress in studying the recognition of minor antigens by CTL has been limited, however, by several observations. First, CTL recognize minor alloantigens only in association with self-class I antigens, not as free antigen. Thus, only target cells which share certain HLA determinants with the CTL can be tested. Second, anti-minor CTL usually can only be generated with cells from in vivo immunized responders. Finally, very little is known about the molecular nature of minor antigens. We have raised CTL lines which distinguish between members of certain HLA-identical sib pairs based on the expression of minor alloantigens. However, as CTL directed against minor alloantigens recognize these determinants only in the context of certain self HLA antigens, called restriction determinants, studies designed to detect minor alloantigens are limited to those target cells which express the restriction determinant of the CTL. Minor antigens on other cells are undetectable. Furthermore, unless a given cell expresses the restriction determinants with which two different CTL lines recognize minor antigens, it is not possible to determine whether the minor alloantigen seen by CTL 1 together with HLA-X is the same or different than the minor alloantigen seen by CTL 2 together with HLA-Y. In this project we seek to circumvent these problems. First, we will integrate plasma membranes containing the appropriate HLA antigens into potential target cells which do not express these specificities naturally. This will be accomplished using the technique of reconstituted Sendai virus envelope-mediated membrane transfer, there-by rendering the cell susceptible to lysis by the CTL line if the minor antigen is expressed. The resulting target cells could then be tested for the expression of minor antigens using all available CTL lines. This will enable studies of the population genetics of these determinants to be undertaken, as well as their systematization. Finally, we will generate anti-minor CTL in a totally in vitro system to ensure a plentiful supply of anti-minor CTL lines.